This proposal merges developmental and chemical biology, computational analysis, and bioengineering to tackle one of the most significant challenges in hematology research-the in vitro derivation of long-term engraftable hematopoietic stem cells from pluripotent stem cells. To date, considerable efforts at directed differentiation o hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have proven only partially successful in mice and largely unsuccessful in humans. The failure to generate true ESC/iPSC-derived HSCs remains a barrier for exploiting patient-derived iPSCs for modeling blood diseases, and precludes harnessing this potentially transformative technology for therapy. This renewal application will build upon progress made during the first two years of the prior grant period to address the central question: What are the developmental pathways that drive the formation of HSCs in embryos, and how can we exploit this knowledge to direct the differentiation of pluripotent stem cells into clinically relevant hematopoietic lineages? We will apply computational algorithms to enhanced expression datasets generated by single cell RNA-seq of hematopoietic stem and progenitor populations from fish, mouse, and human. We will probe gene regulatory networks that direct hematopoietic and lymphoid development, and test candidate transcription factors, non-coding RNAs, and morphogens hypothesized to govern these networks, alone and in combination, through gain and loss-of-function strategies. In a second thrust, we will perform screens in zebrafish blastomeres and human pluripotent stem cells to discover novel chemical and biological regulators of HSCs, and will incorporate chemicals into cell differentiation protocols to drive HSC commitment and enhance HSC function. Lastly, using bioengineered platforms which mimic the biomechanical forces that act on the embryonic hemogenic endothelium, we will configure hemogenic endothelium-on-a-chip to interrogate the effectors and downstream signaling pathways that promote emergence of HSCs and expansion of hematopoietic stem and progenitor cells. Our efforts at defining genetic, chemical and biomechanical mechanisms in hematopoietic development represent complementary as well as synergistic strategies to solve our key challenge of deriving HSCs in vitro.